Non-invasive diagnostic method for the evaluation of intestinal lactase deficiency (hypolactasia)

ABSTRACT

The test of the invention comprises the measuring the total amount of xylose in urine and/or its concentration in blood following oral administration of 4-Oβ-D-galactopyranosyl-D-xylose (4-GX) to the patient. It is a non-invasive test that is based on the direct evaluation of the global enzyme activity in the whole individual, not on measuring the metabolic consequences derived from its deficiency. It does not require specialised equipment, does not cause apparent discomfort in patients with lactase deficiency and is very reliable, thus overcoming the drawbacks of the diagnostic tests currently in use and is a statistically significantly better test in terms of its reliability; consequently it should become the reference or gold standard test for the indication of hypolactasia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/433,002 for “Non-InvasiveDiagnostic Method for the Evaluation of Intestinal Lactase Deficiency(Hypolactasia)” filed on Jan. 14, 2011 in the name of Juan Jose ARAGONREYES et al., which is hereby incorporated herein in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention is included in the pharmaceutical sector and is applicablein the medical diagnostic sector for the evaluation of intestinallactase activity, specifically in intestinal lactase deficiency(hypolactasia) in humans as a non-invasive test of the activity of thisenzyme.

STATE OF THE ART

Intestinal lactase is the enzyme responsible for lactose digestion.Lactase is located in the microvilli, which constitute the so-calledbrush border of the enterocytes of the small intestine, it is anintegral protein of the cell membrane with its active centre directedtowards the lumen of the small intestine, and its activity varies alongthe length of the intestine, the highest activity being found in themiddle jejunum (Newcomer, A. D. et al., 1966; Gastroenterology 51,481-88; Triadou, N. et al., 1983; Gastroenterology 85, 1326-32). Lactoseis not absorbed like other disaccharides through the intestinal mucosabut must be hydrolysed by lactase into its components of galactose andglucose, which are then absorbed, this enzyme accounting for allintestinal lactase activity (Semenza, G. et al., 2001. The Metabolic andMolecular Bases of Inherited Disease. McGraw-Hill. Vol I, pp 1623-1650).The hydrolysis of this disaccharide is the rate limiting step in thedigestion and use of lactose (Dawson, D. J. et al., 1986; Gut 27,521-27).

If there is a lack of lactase, lactose is not hydrolysed in the smallintestine inducing a reduction in gastric emptying speed, increase inintestinal transit time, increase in osmotic pressure and retention offluid in the intestinal cavity, giving rise to bacterial fermentation ofthis fluid in the colon and to the production of gasses such ashydrogen, methane and carbon dioxide and to the formation of short chainfatty acids. As a consequence of these effects, the digestion process isreduced and absorption of monosaccharides falls, giving rise to pain andabdominal cramps, flatulence, audible intestinal noise and diarrhoea. Inthe adult, this set of symptoms is known as hypolactasia or lactoseintolerance and is a very common genetic disorder, affecting to morethan a half of the human species. In the newborn, congenital deficiencyof the enzyme prevents proper utilisation of lactose resulting in severedisorders such as intense diarrhoea and dehydration, derived both fromthe reduction of energy input and the intestinal accumulation ofnon-hydrolysed disaccharide, which requires early detection and a changein diet to lactose-free milk. Lactase deficiency also occurs in asecondary way in a significant number of intestinal pathologies that areaccompanied by various degrees of degradation of the intestinal mucosaincluding celiac disease, inflammatory chronic intestinal disease(Crohn's disease and ulcerative colitis), irritable bowel syndrome,intestinal resection, cystic fibrosis, premature infants, chemotherapytreatments or as an additional disorder of old age. The evaluation oflactase activity is therefore of particular interest ingastroenterology, paediatrics and generally in pathological processeswhere functional integrity of intestinal mucosa or a differentialdiagnosis with the deficiency of this enzyme must be assessed.

Two classes of methodologies are used for the diagnosis of intestinallactase deficiency or lactose intolerance, which have a series ofdisadvantages that in general imply poor reliability and seriousdiscomfort for patients and also require specialised equipment:

-   -   Direct determination of lactase activity in a sample of        intestinal mucosa obtained by biopsy using endoscopy        (Newcomer, A. D. et al., 1966; Gastroenterology 51, 481-88,        Semenza, G. et al, 2001. The Metabolic and Molecular Bases of        Inherited Disease. McGraw-Hill. Vol I, pp 1623-1650; Arola, H.,        1994; Scand. J. Gastroenterol. 202 (29Suppl), 22-35). This is an        invasive method, which only indicates enzyme activity in a        specific part or area of the intestine, which is generally not        the area of highest occurrence of the enzyme (middle jejunum),        as it is very difficult to take biopsies in this area. The        result varies from one sample to another, therefore does not        provide information about the total enzyme activity in an        individual. A poor correlation has been observed between        clinical symptoms of lactose intolerance and lactase activity        measured in intestinal mucosa.    -   Indirect determination, evaluating the metabolic consequences of        enzyme deficiency after the oral lactose overload test        (administration of 1-2 g of lactose per kg body weight to a        maximum of 50 g) such as the appearance of symptoms in the        subject (abdominal pain, flatulence, diarrhoea, etc.), the        determination either in blood or urine of glucose or galactose        levels (McGill, D. B. et al., 1967; Gastroenterology 53, 371-74;        Newcomer, A. D. et al., 1975; N. Engl. J. Med. 293, 1232-36),        determination of the products of lactose hydrolysis by lactase,        either by measurement of gases in the breath such as, for        example, H₂ (Arola, H., 1994. Scand. J. Gastroenterol. 202        (29Suppl), 22-35; Newcomer, A. D. et al., 1975; N. Engl. J. Med.        293, 1232-36; Levitt, M. D., 1969; N. Engl. J. Med. 281, 122-27;        Metz, G. et al., 1975; Lancet 1, 1155-57) or exhaled CO₂        (Newcomer, A. D. et al., 1975; N. Engl. J. Med. 293, 1232-36;        Koetse, H. A. et al., 1999; Test. Scand. J. Gastroenterol. 34,        35-40; Sasaki, Y. et al., 1970; J. Lab. Clin. Med. 76, 824-35).        The main problems with these types of indirect test derive from        the significant degree of accompanying digestive discomfort, as        it is necessary to administer an oral overload of lactose in        order to carry them out, this being particularly severe in        infants. Also, as these are indirect tests, they do not enable        the evaluation of total enzyme activity in an individual but        depend on endogenous production capacity of gases such as H₂ and        CO₂ by the subject. In addition to being variable from one        individual to another, this is affected by various factors        beyond the quantity or activity of lactase enzyme such as        smoking habit, type of diet and prior exercise, emotional state,        diabetes, use of antibiotics, etc.; so there is a high        proportion of false positives and false negatives resulting in        poor test reliability. Various studies have demonstrated poor        correlation between clinical symptoms of lactose intolerance and        the data provided by these types of tests (Davidson, G. P. et        al., 1985; J. Pediatr. Gastroenterol. Nutr. 4, 381-87;        Lifshitz, C. H. et al., 1985; J. Pediatr. Gastroenterol. Nutr.        4, 942-94845, 46).

In summary, indirect methods, although non-invasive, suffer from threefundamental problems:

i) serious accompanying discomfort to patients with lactase deficiencydue to the high lactose doses that must be ingested in all cases;

ii) need for special and unusual equipment and which is not alwaysavailable in all health centres;

iii) relative frequency of false positives and false negatives.

These drawbacks result in diagnoses of lactase deficiency being mademuch less frequently than the high level of incidence in the populationwould lead one to expect.

For these reasons, new diagnostic methods involving little discomfort,simplicity of application and greater reliability are being developed.These are methods for the evaluation of intestinal lactase activitybased on the use of specific disaccharides, structural analogues oflactose, and which can function as substrates for the enzyme, and whichonce ingested, are transformed by the action of intestinal lactase intocertain monosaccharides that are absorbed by intestinal mucosa and canbe determined in the blood or urine.

Spanish patents ES478590 and ES482073 disclose methods based on theevaluation of intestinal lactase activity by oral administration of3-O-methyl lactose, a structural analogue of lactose, and thedetermination of 3-O-methyl-D-glucose in the urine. But these types ofanalogues have not been used in clinical practice as they imply theabsorption into the bloodstream of a non-physiological compound, such as3-O-methyl-D-glucose, and require gas chromatography or high pressureliquid chromatography systems for the determination.

Spanish patent ES 2023556 discloses the preparation of the disaccharide,4-O-β-galactopyranosyl-D-xylose (4-GX), for the evaluation of intestinallactase activity. This disaccharide is administered orally and acts as asubstrate of intestinal lactase, being hydrolysed in the intestinaltract into xylose and galactose. Both compounds are absorbed and asubstantial part of xylose is eliminated in the urine where it can bedirectly determined by a simple colorimetric method. The quantity ofxylose excreted in the urine is correlated with the levels of intestinallactase.

Spanish patent ES 2100131 describes enzymatic processes for thepreparation of mixtures of galactopyranosyl-xylose disaccharidescontaining 4-GX and its regioisomers, 2-O-β-galactopyranosyl-D-xyloseand 3-O-β-galactopyranosyl-D-xylose, and the use of a mixture of thesethree regioisomers in the evaluation of intestinal lactase activity insuckling rats of 12 to 30 days of age. A dose of 16.2 mg of theabove-mentioned mixture was administered and urine was collected for thenext 5 hours, determining therein the eliminated xylose by colorimetricanalysis based on reaction with phloroglucinol and using basal urine asthe blank. A group of these animals was later sacrificed and the lactaseactivity in the intestinal mucosa was determined directly. Theexperiment was repeated in the other animals at days 15, 18, 21, 24 and30. This specification demonstrated that the elimination of xylose inthe urine after oral administration of the mixture to animals duringtheir development phase was proportional to intestinal lactase activitydetermined post-mortem in the intestinal mucosa of these same animals.

Similarly, Spanish patent ES 2182703 discloses an enzymatic process forobtaining 4-GX that involves an enzymatic reaction between D-xylose anda substrate, B-D-xylopyranoside, and a subsequent phase of isolation andpurification of 4-GX. The use of 4-GX in the preparation of compositionsand solutions that are useful for the evaluation of intestinal lactasein humans is also described.

4-GX functions as a structural analogue of lactose, the physiologicalsubstrate of lactase, so that after its oral administration it ishydrolysed by the intestinal mucosa enzyme, passing the reactionproducts into the blood, and one of them, xylose, also appears in theurine. In said biological fluid, its concentration can be evaluated by asimple colorimetric determination based on reaction with phloroglucinol.Compared with the methods used in the state of the art for the diagnosisof intestinal lactase deficiency, this method has the followingadvantages:

-   -   High reliability, as this method is based on the direct        evaluation of enzyme activity through the determination of        xylose in urine or blood (plasma), not on the evaluation of        metabolic consequences deriving from its deficiency. The        information provided is indicative of total lactase activity in        the individual.    -   High sensitivity: the lower detection limit of xylose when        determined by colorimetric analysis based on reaction with        phloroglucinol is 0.1 μg.    -   Simplicity of the required equipment, routinely available in any        heath centre with minimal clinical biochemistry laboratory        facilities, as the evaluation of the enzyme level is only based        on xylose determination, which can be performed by a simple        colorimetric test. Furthermore, this technique does not require        training or development of new analysis methods as xylose        determination is a routine analytical technique in the clinic.    -   Avoidance of discomfort or adverse gastrointestinal effect in        patients with lactase deficiency while carrying out the test.        The oral dose of 4-GX administered is very low, since the        sensitivity of xylose determination is very high. It should also        be pointed out that the hydrolysis of 4-GX by intestinal lactase        results in the appearance of two physiological products,        galactose and xylose, which are eventually absorbed by the        intestinal mucosa. The method does not demand active        participation by the subject, so is of particular interest in        infants. There is no need to administer compounds labelled with        radioactive isotopes.

These studies performed with 4-GX for the evaluation of intestinallactase demonstrate that this is a harmless, rapid and very simple testthat requires minimal equipment and can be highly reliable as it dependsdirectly on the total lactase activity of the individual. Therefore theobject of the present invention is to define the optimum doses and timesfor the evaluation of intestinal lactase activity in humans usingnon-invasive processes by the oral administration of a structuralanalogue of lactose, 4-GX, with this test having the potential to becomethe reference or gold standard test for the determination of intestinallactase deficiency.

Spanish patent ES 2208099 discloses the use of 4-GX in humans for theevaluation of intestinal lactase as a non-invasive diagnostic test forthe deficiency of this enzyme. This patent demonstrates that theexcretion of xylose in urine of healthy lactose-tolerant adultvolunteers over a period of 8 hours following the administration of thedisaccharide increased with the quantity of 4-GX ingested when this wasadministered at doses of 0.25, 0.5, 1.0 and 3.0 g. This demonstratesthat the elimination of xylose by human subjects is dependent on thedose of 4-GX administered, with a dose of 0.25 g of 4-GX beingsufficient to enable reliable detection of xylose in urine after theingestion of the compound. The administration of this dose of 4-GX toadult volunteers with lactose intolerance, who have intestinal lactasedeficiency, gives rise to a marked reduction in the elimination ofxylose in urine compared to the average values observed inlactose-tolerant volunteers, and depended on the degree of enzymedeficiency. This demonstrates that the method presented in the patentenables the evaluation of intestinal lactase activity and is a validtest for non-invasive diagnosis of the deficiency of this enzyme inhumans. This same patent showed that oral administration of 3 g of 4-GXto lactose-tolerant adult volunteers gave rise to the gradual appearanceof xylose in blood plasma samples of these volunteers, determinedcolorimetrically with phloroglucinol. The gradual appearance of xylosein plasma reached a maximum around 2 hours after the ingestion of thedisaccharide. Oral administration of the same quantity of 4-GX to alactose-intolerant adult volunteer diagnosed as hypolactasic resulted ina markedly reduced amount of xylose determined in blood plasma in thissubject at 2 hours following ingestion of the disaccharide. This patenttherefore demonstrates that the determination of xylose in bloodfollowing oral administration of 4-GX enables the evaluation ofintestinal lactase activity and may also be a valid test fornon-invasive diagnosis of the deficiency of this enzyme in humans. Theabove mentioned patent therefore concludes that, taking into account thepattern of xylose appearance in blood and urine with time following oraladministration of 4-GX to lactose-tolerant and lactose-intolerantvolunteers and the high sensitivity of the method used for colorimetricevaluation of xylose with phloroglucinol, the samples of body fluids tobe used with this method in adult subjects can be basal urine prior toingestion of 4-GX and total urine collected for 3 or 4 hours afteringestion of this compound, or a basal blood sample prior to ingestionof 4-GX and another blood sample extracted 2 hours after theadministration of that compound.

However, one of the remaining problems in the state of the art is todetermine the reference values beyond which a subject analysed could beconsidered to present lactose intolerance. The test disclosed in ES2208099 also shows long analysis times of over 2 hours, which would beexcessive for patients, including babies, who have been fasting for atleast 8 hours before performing the test. Moreover, this time are alsoexcessive for processing a high number of samples.

In this sense, the present invention describes the doses and times usedfor the evaluation of intestinal lactase activity in humans bynon-invasive processes through the oral administration of a structuralanalogue of lactose, 4-GX, with the determination of normal limits orcut-off points obtained for the concentration of xylose in blood andurine. Lower values of xylose compared to the normal limits described inthe present invention are considered as positive, i.e. as indicative ofintestinal lactase deficiency, the cause of clinical symptoms of lactoseintolerance. This patent concludes that the optimum dose of 4-GX to beadministered to correctly diagnose (the term correctly diagnose beingunderstood to mean performing a diagnostic test in which subjectsdeficient in intestinal lactase are distinguished from normal subjectswith high reliability) of the degree of hypolactasia is 0.5 g and thetime required for urine collection is not less than 4 hours and notgreater than 5 hours in order to ensure a sufficient quantity ofexcreted xylose. The lowest does of 4-GX to be administered to correctlydiagnose the degree of hypolactasia using xylose concentration in plasmais 3 g, with the sample of plasma to be obtained 90 minutes after theoral administration of the disaccharide. In patent ES 2208099, themeasurement sample was taken at 120 minutes following administration of4-GX, the present invention thus demonstrating surprisingly that theoptimum time for sampling is approximately 25% shorter, whichadditionally enables more determinations or diagnostic tests to becarried out using the method described in the present invention, moreimportantly with higher levels of reliability.

The doses of 4-GX to be administered, the times for measuring theconcentration of xylose both in blood and urine samples and thedetermination of the normal ranges obtained in healthy volunteers forthe diagnosis of intestinal lactase deficiency by the administration ofvarious doses of 4-GX make this a new “gold standard” test for thediagnosis of intestinal lactase activity by non-invasive methods, whichdid not exist to date. The reference or gold standard test until thepresent invention was taken to be the biopsy test, which, as previouslydescribed, is an invasive test and only indicates the activity of theenzyme in a particular part of the intestine from which the biopsy istaken, which additionally does not usually coincide with the area ofmaximum enzyme levels.

As can be seen in the present invention, no clinically significantadverse reactions were found at any of the doses of 4-GX used, all ofthem showing a similar safety and tolerance profile.

DESCRIPTION OF THE INVENTION Brief Description of the Invention

The present invention describes the sensitivity, specificity, positivepredictive value and negative predictive value of the non-invasive testof the activity of intestinal lactase based on oral administration of4-GX and the subsequent determination in urine and/or blood of itshydrolysis product, xylose.

One of the main objectives of the present invention has been toestablish the lower limits of xylose concentration in healthyindividuals using the tests with 4-GX on blood and/or urine described inthe invention. Thus individuals presenting lower values of xylosecompared to the normal limits described in the present invention areconsidered as individuals with intestinal lactase deficiency, the causeof clinical symptoms of lactose intolerance. The results obtained usingthe doses and times described in the present invention are compared withthose obtained by tests used in clinical practice to date: samples ofintestinal biopsy, hydrogen breath test and the capillary-blood glucosetest.

By means of the statistical data shown in the present inventionregarding the optimum doses and times used for the tests described inthe present invention compared to data obtained by the other testsperformed and that are part of the state of the art, it is concludedthat the test of measuring intestinal lactase activity by theadministration of 4-GX, described in the present invention, beconsidered to be the reference or gold standard test for the diagnosisof hypolactasia, since in addition to being a non-invasive test it iscapable of distinguishing false positives and false negatives thatappear in the tests performed using the older reference method:measurement of intestinal lactase in biopsy, taking as a comparison thenormal limit used in clinical practice for this diagnostic test. Thesecharacteristics, along with the absence of adverse reactions inintolerant patients and the simplicity of carrying out the test, makethe tests described in the present invention into an optimum functionaldiagnostic for the diagnosis of lactase intolerance.

Obtaining the above mentioned statistical data on sensitivity,specificity, positive predictive value and negative predictive valuewere carried out following the administration of a single oral dose of 3g of 4-GX for the determination of the maximum concentration of xylosein plasma and 0.5 g of 4-GX for the determination of the total amount ofxylose excreted in urine. The concentration of xylose in the blood(plasma) was measured 90 minutes after ingestion of 4-GX and the amountof xylose in urine was measured in the total urine collected for up to 4hours following the administration of the above indicated dose of 4-GX(0-4-hour urine) and/or in the total urine collected up to 5 hours afterthe administration of the above indicated dose of 4-GX (0-5-hour urine).

For the effects of the present invention, the term gold standard test orreference test is a diagnostic test that is considered to be definitivefor diagnosing a disease in an individual; in the case of the presentinvention, the non-invasive test for the diagnosis of intestinal lactasedeficiency with 4-GX under the conditions described (administeredamounts and times of measurement) is considered to be the gold standardtest or reference test for the diagnosis of hypolactasia, using eitherplasma or urine.

For the effects of the present specification, the terms: referencethreshold value, normal limit, lower limit of normal, cut-off point andcut-off are synonymous, all of them being defined as those values belowwhich a patient is diagnosed as hypolactasic by the 4-GX test describedin the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Percentages of sensitivity (A), specificity (B), positivepredictive value (C), negative predictive value (D), positive likelihoodratio (E) and negative likelihood ratio (F) of the various testsperformed in patients (n=205) with symptoms suggestive of lactoseintolerance after the administration of different doses of 4-GX comparedto the determination of intestinal lactase activity in biopsy taken asthe gold standard test.

FIG. 2. ROC curves (“Receiver-Operating-Characteristic”, indicating theaccuracy of a diagnostic test) of the various tests performed inpatients (n=205) with symptoms suggestive of lactose intolerance afterthe administration of different doses of 4-GX compared to thedetermination of intestinal lactase activity in biopsy taken as the goldstandard.

FIG. 3. Percentages of sensitivity (A), specificity (B), positivepredictive value (C), negative predictive value (D), positive likelihoodratio (E) and negative likelihood ratio (F) of the various testsperformed in patients (n=205) with symptoms suggestive of lactoseintolerance after the administration of various doses of 4-GX comparedto the determination of intestinal lactase activity in biopsy taken asthe gold standard test after recalculating the statistical parameterstaking into account the false positive values for the patients includedin Table 9.

FIG. 4. ROC curves of the various tests performed on patients withsymptoms suggestive of lactose intolerance after the administration ofvarious doses of 4-GX compared to the determination of intestinallactase activity in biopsy taken as the gold standard test afterrecalculating the statistical parameters taking into account the falsepositive values and false negative values for the patients included inTable 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a non-invasive diagnostic method forintestinal lactase deficiency comprising the stages of:

-   -   a) Administering via the oral route to the individual who is the        object of the test a quantity of 4-GX of between 0.125 g and 6 g        after a fasting period of at least 8 hours.    -   b) Collecting total excreted urine by the individual between the        time of administration of 4-GX and at least up to 4 hours        following this administration.    -   c) Determining in vitro the total amount of xylose excreted in        the urine collected in this period of at least 4 hours following        administration of 4-GX.    -   d) Comparing the value obtained in step c) with a threshold        reference value obtained in vitro from a population of healthy        control individuals subjected to the same protocol, below which        threshold value the subject is considered to be suffering from        intestinal lactase deficiency.

In a preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that instep b) the urine is collected over a period of at least 4 hours or atleast up to 5 hours following administration of 4-GX.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thedoses of 4-GX administered are selected from: 0.125 g, 0.250 g, 0.5 g, 1g, 3 g and 6 g; the preferred dose to be administered being that of 0.5g of 4-GX. It should be appreciated that the 4-GX doses can includesmall quantities of water which remain subsequent to the drying processof the 4-GX product. For example, the 4-GX product can contain about 8wt % to about 12 wt %, most likely about 10 wt % water, based on thetotal weight of the product. Accordingly, the aforementioned 4-GX dosesadministered would actually include 0.1125 g, 0.225 g, 0.45 g, 0.9 g,2.7 g and 5.4 g of actual 4-GX product, respectively, if they contain 10wt % water.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 0.125 g of 4-GX(or 0.1125 g in a 4-GX sample including 10 wt % water) is 11.88 mg andin 5 h urine it is 16.72 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 0.250 g of 4-GX(or 0.225 g in a 4-GX sample including 10 wt % water) is 20.62 mg and in5 h urine it is 28.08 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 0.5 g of 4-GX (or0.45 g in a 4-GX sample including 10 wt % water) is 27.58 mg and in 5 hurine it is 37.87 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 1 g of 4-GX (or0.9 g in a 4-GX sample including 10 wt % water) is 33.04 mg and in 5 hurine it is 41.35 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 3 g of 4-GX (or2.7 g in a 4-GX sample including 10 wt % water) is 45.58 mg and in 5 hurine it is 69.57 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thereference threshold value of the total quantity of xylose in urine at 4h after the administration to the patient of a dose of 6 g of 4-GX (or5.4 g in a 4-GX sample including 10 wt % water) is 87.96 mg and in 5 hurine it is 110.12 mg.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by urine test is characterised in that thecomparison that takes place in step d) determines the degree ofintestinal lactase deficiency (hypolactasia) in the individual subjectedto the test.

Another object described in the present invention relates to anon-invasive diagnostic method for intestinal lactase deficiencycomprising the stages of:

a) Extracting a blood sample from the individual who is the object ofthe test after fasting for 8 hours.

b) Administering via the oral route to the individual who is the objectof the test a quantity of 4-GX of between 0.125 g and 6 g.

c) Extracting a blood sample from this individual at 90 minutesfollowing administration of 4-GX.

d) Determining in vitro the concentration of xylose in the blood sampleextracted in step a),

e) Determining in vitro the concentration of xylose in the blood sampleextracted in step c).

f) Subtracting the values of in vitro xylose concentrations obtained instep e) from those obtained in step a).

g) Comparing the value obtained in step f) with a threshold referencevalue obtained in vitro from a population of healthy control individualssubjected to the same protocol, below which threshold the subject isconsidered to be suffering from intestinal lactase deficiency.

In a preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thedetermination of the concentration of xylose in the blood is preferablyperformed in plasma.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thedoses of 4-GX administered are selected from: 0.5 g, 1 g, 3 g, and 6 g;the preferred dose to be administered being that of 3 g of 4-GX. Itshould be appreciated that the 4-GX doses can include small quantitiesof water which remain subsequent to the drying process of the 4-GXproduct. For example, the 4-GX product can contain about 8 wt % to about12 wt %, most likely about 10 wt % water, based on the total weight ofthe product. Accordingly, the aforementioned 4-GX doses administeredwould actually include 0.45 g, 0.9 g, 2.7 g and 5.4 g of actual 4-GXproduct, respectively, if they contain 10 wt % water.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thereference threshold value of the concentration of xylose in blood afteradministration to the patient of the dose of 0.5 g of 4-GX (or 0.45 g ina 4-GX sample including 10 wt % water) is 0.41 mg/dL.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thereference threshold value of the concentration of xylose in blood afteradministration to the patient of the dose of 1 g (or 0.9 g in a 4-GXsample including 10 wt % water) of 4-GX is 0.53 mg/dL.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thereference threshold value of the concentration of xylose in blood afteradministration to the patient of the dose of 3 g (or 2.7 g in a 4-GXsample including 10 wt % water) of 4-GX is 0.97 mg/dL.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thereference threshold value of the concentration of xylose in blood afteradministration to the patient of the dose of 6 g of 4-GX (or 5.4 g in a4-GX sample including 10 wt % water) is 1.44 mg/dL.

In another preferred embodiment, the non-invasive diagnostic method forintestinal lactase deficiency by blood test is characterised in that thecomparison that takes place in step g) determines the degree ofintestinal lactase deficiency (hypolactasia) in the individual subjectedto the test.

Another of the objects of the present invention relates to 4-GX to beused as a reference test for the non-invasive diagnosis of intestinallactase deficiency, being administered at doses of between 0.125 g and 6g in a test of the total in vitro quantity of xylose in urine collectedduring a period of at least 4 hours after the administration of 4-GX. Itshould be appreciated that the 4-GX doses can include small quantitiesof water which remain subsequent to the drying process of the 4-GXproduct. For example, the 4-GX product can contain about 8 wt % to about12 wt %, most likely about 10 wt % water, based on the total weight ofthe product. Accordingly, the aforementioned 4-GX doses administeredwould actually be between 0.1125 g and 5.4 g of actual 4-GX product,respectively, if they contain 10 wt % water.

In a preferred embodiment of the invention, 4-GX is used as anon-invasive reference test for the non-invasive diagnosis of intestinallactase deficiency by urine test, the time for the collection of thisurine being at least 5 hours following the administration of 4-GX.

In another preferred embodiment of the invention, 4-GX is used as areference test for the non-invasive diagnosis of intestinal lactasedeficiency by urine test, when it can be administered in any of thedoses selected from: 0.125 g, 0.250 g, 0.5 g, 1 g, 3 g and 6 g (or0.1125 g, 0.225 g, 0.45 g, 0.9 g, 2.7 g, and 5.4 g, respectively, in a4-GX sample including 10 wt % water).

In another preferred embodiment of the invention, 4-GX is used in areference test for the non-invasive diagnosis of intestinal lactasedeficiency by urine test, when it can be preferably administered in adose of 0.5 g (or 0.45 g in a 4-GX sample including 10 wt % water).

In a preferred embodiment of the invention, 4-GX is used in a referencetest for the non-invasive diagnosis of intestinal lactase deficiency byurine test as previously indicated, comparing the value obtained invitro of total xylose excreted in urine with a reference threshold valueobtained in vitro in healthy control individuals subjected to the samereference test with 4-GX, this comparison determining the degree ofintestinal lactase deficiency (hypolactasia).

Another object of the present invention relates to 4-GX to be used as areference test for the non-invasive diagnosis of intestinal lactasedeficiency, being administered at doses of between 0.125 g and 6 g in atest for the in vitro detection of the xylose concentration in bloodobtained 90 minutes after the administration of 4-GX. It should beappreciated that the 4-GX doses can include small quantities of waterwhich remain subsequent to the drying process of the 4-GX product. Forexample, the 4-GX product can contain about 8 wt % to about 12 wt %,most likely about 10 wt % water, based on the total weight of theproduct. Accordingly, the aforementioned 4-GX doses administered wouldactually be between 0.1125 g and 5.4 g of actual 4-GX product,respectively, if they contain 10 wt % water.

In a preferred embodiment of the invention, the determination of theconcentration of xylose using 4-GX in a reference test for thenon-invasive diagnosis of intestinal lactase deficiency is preferablyperformed in plasma.

In another preferred embodiment of the invention, 4-GX is used in areference test for the non-invasive diagnosis of intestinal lactasedeficiency by blood test, when it can be administered in any of thedoses selected from: 0.5 g, 1 g, 3 g and 6 g (or 0.45 g, 0.9 g, 2.7 g,and 5.4 g, respectively, in a 4-GX sample including 10 wt % water).

In another preferred embodiment of the invention, 4-GX is used in areference test in the non-invasive diagnosis of intestinal lactasedeficiency by blood test, when it can be preferably administered in adose of 3 g (or 2.7 g in a 4-GX sample including 10 wt % water).

In a preferred embodiment of the invention, 4-GX is used in a referencetest in the non-invasive diagnosis of intestinal lactase deficiency byblood test as previously indicated, comparing the value obtained invitro of xylose concentration in blood with a reference threshold valueobtained in vitro in healthy control individuals subjected to the samereference test with 4-GX, this comparison determining the degree ofintestinal lactase deficiency (hypolactasia).

The examples detailed below are for the purpose of illustrating theinvention and do not limit its scope.

EXAMPLE 1 Sample Selection

Two groups of subjects were selected. A first group composed of 42healthy controls and a second group composed of 205 subjects with aclinical history suggestive of lactose intolerance. Firstly, the lowestsuitable oral dose of 4-GX administered to healthy volunteers wasdetermined that enabled reliable detection (in terms of accuracy andreproducibility) of xylose levels in urine and blood via the analyticalmethod to be used. In addition, the tolerance of these subjects to thevarious doses of 4-GX used in the test and the pharmacokinetics for eachdose administered were analysed, both in urine and blood. The doses of4-GX used were: 0.125 g, 0.250 g, 0.5 g, 1 g, 3 g and 6 g in addition toplacebo in 12 healthy volunteers with washout period between doses ofbetween three and seven days. It should be appreciated that the 4-GXdoses can include small quantities of water which remain subsequent tothe drying process of the 4-GX product. For example, the 4-GX productcan contain about 8 wt % to about 12 wt %, most likely about 10 wt %water, based on the total weight of the product. Accordingly, theaforementioned 4-GX doses administered would actually include 0.1125 g,0.225 g, 0.45 g, 0.9 g, 2.7 g and 5.4 g of actual 4-GX product,respectively, if they contain 10 wt % water. The study was performedwith seven treatment periods and a washout time between the variousperiods of at least 3 days. In each study period, an ascending dose of4-GX was administered and the serum and urine concentrations of xylosewere measured at various times up to 8 hours following theadministration of the drug.

The main measured variable was the quantity of xylose in urine collectedfollowing oral administration of 4-GX at the following times: basal, 0-1h, 1-2 h, 2-3 h, 3-4 h, 4-5 h, 5-6 h, 6-7 h and 7-8 h. Secondaryvariables quantified were the plasma concentration of xylose measured inblood samples extracted following the oral administration of 4-GX at thefollowing times: basal, 30 min, 60 min, 90 min, 120 min, 150 min, 3 h, 4h, 5 h, 6 h, 7 h and 8 h. A pharmacokinetic analysis was also performedusing the values of AUC (area under the curve of variation in the plasmaxylose concentration against time), Cmax (maximum xylose plasmaconcentration), Tmax (time at which maximum xylose plasma concentrationwas reached) and T₁₁₂ (elimination half-life), calculated from theplasma concentrations of xylose derived from the hydrolysis of 4-GX byintestinal lactase.

The data obtained in the test by the administration of 4-GX in the groupof healthy controls was used to determine the optimum doses and timesfor determining if a patient showed lactose intolerance by the methoddescribed in the present invention and also the lower limits of normalof xylose in urine and blood (cut-off points) for each of the dosesused. The indicator of the lower limit of normal was taken to be thedifference between the average xylose concentrations in blood and/or thedifference between the average total amounts of xylose excreted in theurine, and 1.96 times the standard deviation.

Once the normal lower limits of xylose in healthy subjects had beenobtained, 205 subjects with a clinical history suggestive of lactoseintolerance were subjected to the various diagnostic tests for lactoseintolerance in the state of the art in order to compare with the resultsobtained by the diagnostic test of lactose intolerance by the oraladministration of 4-GX. The diagnostic tests to which these patientswere subjected were:

-   -   Intestinal biopsy: determining intestinal lactase activity        therein.    -   Hydrogen breath test after the oral administration to patients        of 50 g of lactose. While this test was being carried out,        several capillary-blood glucose test (Glycemia tests) were        carried out, calculating the highest increase observed in the        blood glucose concentration compared to basal glucose level        before starting the test.    -   4-GX test in total urine at 0-4 and 0-5 hours, which was orally        administered to patients 0.5 g of this product (or 0.45 g in a        4-GX sample including 10 wt % water), collecting the total urine        accumulated over 0-4 h and 0-5 h following the administration of        4-GX and determining the amount of excreted xylose in both        volumes of urine.    -   4-GX test in blood which was orally administered to patients 3 g        of this product (or 2.7 g in a 4-GX sample including 10 wt %        water) to determine the concentration of plasma xylose in a        blood sample 90 minutes after ingestion of the product. The        concentration obtained was compared to a basal blood sample        extracted before ingestion of 4-GX.

The 4-GX was orally administered in a single dose dissolved in 100 ml ofwater. To demonstrate the effectiveness, specificity and sensitivity ofthe intestinal lactase test by the administration of the above indicateddoses of 4-GX in subjects with a clinical history suggestive of lactoseintolerance, the results obtained via these tests were compared with theresults obtained with the other tests: Biopsy, hydrogen breath test andcapillary-blood glucose test after a lactose overload.

EXAMPLE 2 Selection of the Optimum Dose and Time for the Diagnosis ofLactose Intolerance by the Oral Administration of 4-GX

The main pharmacokinetic parameters that were evaluated for urineexcretion of xylose were the maximum rate of urinary excretion (U ratemax) and the total quantity of xylose excreted over the period ofobservation (Ae 0-t); using the amounts of xylose excreted in each urinesample collection period, the quantities accumulated for each intervalwere calculated so that it was possible to determine the minimum urinecollection time that would distinguish accumulated excretion at thevarious doses compared to placebo (Table 1).

The statistical analysis of the pharmacokinetic parameters of urinaryxylose excretion showed that all the doses of 4-GX, in terms of themaximum urinary excretion rate (U rate max) and accumulated excretionover 8 hours of duration of the observation period (Ae 0-t) weresignificantly different from that obtained with placebo. The analysis ofthe quantities accumulated over the observation period for the variousdoses showed that the lowest dose of 4-GX for which significantdifferences in total xylose excretion compared to placebo could bedetermined was the dose of 0.5 g (or 0.45 g in a 4-GX sample including10 wt % water) (amount accumulated at 8 hours was 25.52 mg; CI9.82-41.82) and that this difference could be observed in the urinecollection period of between 3 and 4 hours (amount accumulated at 4hours was 57.24 mg; IC95%: 46.66-67.82) (Table 2) and was still moreevident in the interval between 4 and 5 hours (amount accumulated at 5hours was 65.05 mg; IC95%; 53.81-73.04) (Table 2). Therefore, it can beconcluded that significant differences in the accumulated xyloseexcreted, compared to placebo, can be detected by measuring theaccumulated excretion in urine at least 4 hours after administration ofa dose of 0.5 g of 4-GX (or 0.45 g in a 4-GX sample including 10 wt %water).

TABLE 1 Summary of kinetic parameters of xylose concentration excretedin urine at different 4-GX doses. XYLOSE Placebo 0.125 g 0.250 g 0.5 g 1g 3 g 6 g U rate Max 4.49 9.14 14.01 18.67 26.69 52.02 82.81 (mg/h) *(2.04) (2.90) (3.82) (5.96) (7.56) (21.83) (30.46) Ae 0-t * 18.34 41.4563.97 80.15 112.32 207.82 315.74 (mg/dl/h) (7.67) (7.52) (13.91) (19.65)(38.79) (95.89) (96.71) Tmax U 0.5 2.0 1.5 1.5 2.5 1.5 2.0 rate (h) **(0.5-5.5) (0.5-7.5) (0.5-2.5) (0.5-2.5) (1.5-2.5) (1.5-3.5) (1.5-2.5)T1/2 (h) * 3.35 3.53 3.77 2.50 2.43 1.93 3.06 (1.90) (1.59) (2.09)(1.49) (0.44) (0.77) (1.90) * Expressed as mean (standard deviation) **Expressed as median (range) U rate Max (mg/h): Maximum urinary excretionrate Ae 0-t (mg/dL/h): accumulated xylose excretion over the 8 hoursduration of the observation period. Tmax U rate (h): time to reach themaximum xylose concentration excreted in urine T1/2 (h): xyloseelimination half-life in urine

TABLE 2 Average amount (mg) of xylose accumulated in urine in each urinecollection period for each administered dose. The 95% confidenceinterval (CI 95%) is shown. Dose Time (mg) 0-1 h 1-2 h 2-3 h 3-4 h 4-5 h5-6 h 6-7 h 7-8 h 0 Mean 3.90 6.20 8.68 11.41 14.47 17.91 21.67 25.82lower CI 2.29 4.17 6.37 8.46 10.16 11.17 11.02 9.82 upper CI 5.51 8.2310.99 14.37 18.78 24.65 32.31 41.82 125 Mean 6.81 12.80 19.29 25.6230.45 34.51 38.34 41.45 lower CI 5.01 10.54 16.51 21.48 26.14 30.0633.42 36.67 upper CI 8.62 15.06 22.07 29.77 34.76 38.96 43.26 46.23 250Mean 8.44 20.59 32.48 41.03 48.37 54.49 59.68 63.97 lower CI 6.45 16.9327.38 34.05 40.89 46.44 51.07 55.13 upper CI 10.44 24.24 37.57 48.0155.84 62.54 68.29 72.81 500 Mean 13.47 31.13 46.37 57.24 65.05 71.2375.86 80.15 lower CI 10.48 24.69 37.09 46.66 53.81 59.39 63.89 67.66upper CI 16.45 37.56 55.64 67.82 76.29 83.06 87.83 92.64 1000 Mean 17.4941.24 65.04 79.60 92.10 100.55 106.78 112.32 lower CI 12.83 31.36 52.9563.74 73.04 79.11 83.62 87.67 upper CI 22.16 51.11 77.13 95.47 111.16121.99 129.93 136.98 3000 Mean 25.98 70.29 110.98 114.16 168.95 187.57199.17 207.82 lower CI 15.09 43.40 74.64 100.74 119.18 131.69 141.10146.88 upper CI 36.87 97.18 147.33 187.59 218.71 243.45 257.24 268.766000 Mean 30.04 99.36 173.64 228.07 266.89 285.99 304.94 315.74 lower CI19.25 72.71 136.54 180.90 212.27 228.61 244.60 254.27 upper CI 40.63126.01 210.74 275.24 321.52 343.37 365.28 377.20

The next objective was the determination and pharmacokinetic analysis ofthe xylose concentration in plasma in order to identify the smallestdose of 4-GX that could be distinguished from placebo (without 4-GXadministration) to be used in the 4-GX test for the diagnosis ofintestinal lactase deficiency (Table 3).

For all administered doses, maximum concentrations of plasma xyloseappeared between 1 and 2 hours following oral administration of 4-GX(median 90 minutes). Differences compared to placebo, possiblysignificant from the clinical point of view, started to appear from thedose of 1 g (or 0.9 g in a 4-GX sample including 10 wt % water) (Cmaxfor 1 g was 5.44±0.68 mg/dl; IC90%: 124.63-135.65) (Table 4). Applying aconfidence interval of 95%, more demanding and therefore more precise,these observed differences compared to placebo reduced slightly, beingfeasible that the dose of 1 g was not sufficient to obtaindiscriminatory power in almost all cases to be used as a diagnostic tool(CI95%: 123.59-136.78). The dose of 3 g (or 2.7 g in a 4-GX sampleincluding 10 wt % water) showed a Cmax of 6.08 ±0.73 mg/dl, and the 90%confidence interval compared to placebo showed better discriminatorypower (CI90%: 139.36-151.38) (Table 4), which was also maintained aftercalculating the 95% confidence interval (CI95%: 138.20-152.95), forwhich reason it seemed more reasonable to select this dose forsubsequent clinical development of 4-GX as a diagnostic test for lactasedeficiency.

TABLE 3 Summary of kinetic parameters of xylose concentration in bloodat different 4-GX doses. XYLOSE Placebo 0.125 g 0.250 g 0.5 g 1 g 3 g 6gCmax* 4.17 4.58 4.92 5.18 5.44 6.08 7.02 (mg/dl) (0.39) (0.59) (0.50)(0.55) (0.68) (0.73) (0.91) AUC 0-t* 31.77 32.49 34.25 35.53 36.60 39.2441.39 (mg/dl/h) (3.11) (3.43) (3.68) (3.88) (4.99) (4.60) (4.69) Tmax**1.0 1.0 1.5 1.5 1.5 1.5 1.5 (h) (0.0-6.0) (0.5-1.5) (0.5-2.0) (1.5-2.0)(0.5-3.0) (1.0-2.0) (1.5-2.0) T1/2* (h) 38.91 27.28 30.52 24.64 15.317.21 9.64 (28.7) (9.28) (28.44) (14.63) (4.60) (21.59) (3.46)*Expressed as mean (standard deviation) **Expressed as median (range)Cmax: maximum plasma xylose concentration AUC 0-t (mg/dL/h): area underthe curve of variation in the plasma xylose concentration against timeTmax (h): time to achieve the maximum plasma xylose concentration T1/2(h): xylose elimination half-life.

TABLE 4 Summary of the statistical analysis of the concentration ofxylose in plasma compared to placebo following 4-GX administration.VARIABLE Dose RATIO IC^(90%) Ln Cmáx 0.125 g 109.45 104.90 114.18 0.250g 117.91 113.02 123.02 0.5 g 123.95 118.80 129.31 1 g 130.02 124.63135.65 3 g 145.39 139.36 151.68 6 g 167.51 160.56 174.76 Ln AUC_(0-t)0.125 g 102.21 98.55 106.01 0.250 g 107.72 103.86 111.72 0.5 g 111.72107.72 115.87 1 g 114.69 110.59 118.95 3 g 123.27 118.86 127.85 6 g130.08 125.42 134.91 Ln Cmax: Neperian logarithm of the maximum plasmaxylose concentration. Ln AUC_(0-t): Neperian logarithm of the area underthe curve of variation in the plasma xylose concentration against time.

In view of the results, it can be concluded that: the lowest dose of4-GX for which the excretion of xylose in urine can be reliablydistinguished from placebo is 0.5 g (or 0.45 g in a 4-GX sampleincluding 10 wt % water) and the time required for urine collectionshould be at least 4 hours in order to assure collection of a sufficientquantity of excreted xylose. The highest concentrations of xylose inplasma obtained using different doses of 4-GX suggest that the lowestdose for which significant differences compared to placebo can bedistinguished is 3 g (or 2.7 g in a 4-GX sample including 10 wt % water)and the determination of plasma xylose concentration should be at 90minutes following oral administration of 4-GX.

EXAMPLE 3 Determination of the Lower Limits of Normal of the TotalQuantity of Xylose Excreted in Urine and Blood Concentration in HealthyControl Individuals

The data obtained with the 4-GX administration test in the healthycontrol group was used to determine the lower limits of normal of xylosein urine and blood (cut-off points). The cut-off points for the 4-GXtest in urine were obtained for times of 4 hours and 5 hours because, asalready seen in example 2, these times are the best for determining theamount of xylose excreted in urine following 4-GX administration andperforming a reliable diagnosis of lactase deficiency. Similarly, thecut-off points for the determination of the blood xylose concentrationfollowing 4-GX administration were obtained for the time of 90 minutesfollowing ingestion of the disaccharide. The difference between theaverage xylose concentration (in blood and the total quantity of xyloseexcreted in the urine) and 1.96 times the standard deviation was usedfor the lower limit of normal indicator. The results obtained are shownin Table 5. The lower limit of xylose accumulated in 4-hour urine was27.58 mg; xylose accumulated in 5-hour urine was 37.87 mg and theincrease in the concentration of plasma xylose after 90 minutesfollowing ingestion of the product, compared to basal blood, was 0.97mg/dL.

Subjects participating in the study who presented levels of xylose belowthese normal limits were considered as positive, i.e. these levels areindicative of intestinal lactase deficiency, the cause of the clinicalsymptoms of lactose intolerance.

TABLE 5 Cut-off points of the three diagnostic tests for intestinallactase deficiency by the use of different doses of 4-GX. Diagnostictest 4-GX (g) Cut-off point 4-hour urine 0.125 11.88 mg 0.250 20.62 mg0.5 27.58 mg 1 33.04 mg 3 45.58 mg 6 87.96 mg 5-hour urine 0.125 16.72mg 0.250 28.08 mg 0.5 37.87 mg 1 41.35 mg 3 69.75 mg 6 110.12 mg 90-minblood 0.5 0.41 mg/dL 1 0.53 mg/dL 3 0.97 mg/dL 6 1.44 mg/dL

EXAMPLE 4 Determination of the Total Amount of Xylose Excreted in Urinein Subjects with a Clinical History Suggestive of Lactose Intolerance

Having obtained the lower limits of normal or cut-off points for each ofthe doses and the optimum times used in the present invention, the totalamounts of xylose excreted in urine of subjects with a clinical historysuggestive of lactose intolerance were measured. From here on, theoptimum dose of 0.5 g of 4-GX (or 0.45 g in a 4-GX sample including 10wt % water) was used in the 4-GX urine test for the diagnosis ofintestinal lactase deficiency by the non-invasive method described inthe present invention. This dose was dissolved in 100 ml water, andfollowing the test protocol, 2 samples of urine excreted during the 5hours following the administration of the drug were taken, in twointerval groups: at 4 hours following administration of 4-GX and at 5hours following administration of 4-GX.

EXAMPLE 5 Determination of the Blood Xylose Concentration in Subjectswith a Clinical History Suggestive of Lactose Intolerance

In the same way as the case for the determination of the amount ofxylose excreted in urine described in the previous example, for thedetermination of xylose concentrations in blood, 3 g of 4-GX (or 2.7 gin a 4-GX sample including 10 wt % water) dissolved in approximately 100ml water was administered to patients with a clinical history suggestiveof lactose intolerance and to healthy volunteers, and according to thetest protocol, 2 samples of blood were taken at the following times:basal (before the administration of the drug) and at 90 minutesfollowing administration of 4-GX. The blood samples were centrifuged toobtain plasma in which the analysis of the amount of xylose present wasperformed.

EXAMPLE 6 Statistical and Analytical Method

From the data obtained from the various urine tests for intestinallactase deficiency following oral ingestion of 4-GX described in thepresent invention, a descriptive statistical analysis was performed ofthe amount of xylose excreted in the urine up to 4 hours and up to 5hours following said administration. The same was done with the bloodtest for intestinal lactase activity following oral ingestion of 4-GX,where descriptive statistical analysis was performed on the amount ofxylose present in the plasma of subjects at 90 minutes followingadministration of 4-GX. The net increase in the plasma xyloseconcentration compared to basal values was also calculated.

The results were expressed as mean, standard deviation, maximum value,median value and minimum value. The 95% confidence intervals were alsocalculated for each of the analysed variables. The statistical packageSPSS v14.0 (SPSS Inc., Chicago) was used for data analysis.

EXAMPLE 7 Measurement of Intestinal Lactase Activity with the 4-GX TestDescribed in the Invention and Comparison of the Results Obtained Usingthis Test with Those Obtained Using the Intestinal Biopsy Test, HydrogenBreath Test and Capillary-Blood Glucose Test

To obtain the statistical parameters of lactase determination using thevarious 4-GX tests described in the present invention and later tocompare them with the results obtained from the intestinal biopsy test,hydrogen in exhaled air test and capillary-blood glucose test, thenormal limits of the measurement of the total amount of xylose excretedin 4-hour and 5-hour urine and the concentration of blood xylose after90 minutes following ingestion of 4-GX described in Table 5 were used.The normal limits for the other tests were those used in common clinicalpractice for the diagnosis of lactose intolerance (Arola, H. (1994)Diagnosis of hypolactasia and lactose malabsorption. Scand. J.Gastroenterol. 20229 (Suppl), 26-35), which are:

For the measurement of lactase by intestinal biopsy, the lower normallimit is established at 10 units/g protein, with values less than thisbeing considered positive.

For the hydrogen breath test, the normal limit is established at 20 ppm(parts per million) as the maximum peak value compared to the basalvalue during the performance of the test, with values higher than thisbeing considered as positive.

For the capillary-blood glucose test, the normal limit is established asan increase of 25 mg/dL as a maximum peak compared to the basal valueduring the hydrogen breath test.

Taking these normal limits into account, an analysis of intestinallactase deficiency in the 205 patients enrolled in the study wasperformed using the various techniques described above. The resultsobtained are shown in Table 6. As shown in Table 6, the intestinalbiopsy test detected 52.7% of the 205 subjects studied as being positive(those presenting intestinal lactase deficiency), while some 47.3% ofthese subjects had normal levels of the enzyme. The intestinal lactasetest using 4-GX administration and measuring 4-hour urine showed that51.2% of the patients presented intestinal lactase deficiency, whereasthe 4-GX and 5-hour urine test showed that 53.2% of the patients werepositive. Using the 4-GX and blood test, the proportion of patientspresenting intestinal lactase deficiency was 51.7%. The percentages ofpatients presenting intestinal lactase deficiency using the other twotests, the hydrogen breath test and the capillary-blood glucose test,were less than those obtained by the tests mentioned above, that is 45%and 47% respectively.

TABLE 6 Percentages of patients presenting intestinal lactase deficiencyby the various diagnostic tests and applying the normal limits describedabove. No. Positive No. Negative Intestinal biopsy test 108 (53%)  97(47%) 4-GX test in 4-hour urine 105 (51%) 100 (49%) 4-GX test in 5-hoururine 109 (53%)  96 (47%) 4-GX test in 90-minutes blood 106 (52%)  97(48%) Hydrogen breath test  93 (45%) 112 (55%) Capillary-blood glucosetest  96 (47%) 109 (53%)

The tests performed in this study (biopsy, 4-GX and 4-hour urine, 4-GXand 5-hour urine, 4-GX and blood, hydrogen breath test andcapillary-blood glucose test) are for diagnosing intestinal lactasedeficiency or hypolactasia by the measurement of variables that,directly or indirectly, reflect enzyme activity. Therefore the criteriafor positive diagnosis are established exclusively from normal cut-offpoints for each test. The cut-off points are independent of gender, ageand race.

The results obtained in each of the tests performed on these patientsafter analysing the data are shown in the form of statistical parametersin Table 7.

TABLE 7 Statistical parameters calculated after analysing the dataobtained from the different tests performed and considering themeasurement of lactase in intestinal biopsy as the gold standard test ineach case. CI 95% 4-GX test in 4-hour urine Sensitivity (%) 90.74 83.63to 95.47 Specificity (%) 92.78 85.70 to 97.05 Positive predictive value(%) 93.33 86.75 to 97.28 Negative predictive value (%) 90.00 82.38 to95.10 4-GX test in 5-hour urine Sensitivity (%) 93.52 87.10 to 97.35Specificity (%) 91.75 84.39 to 96.37 Positive predictive value (%) 92.6686.05 to 96.78 Negative predictive value (%) 92.71 85.55 to 97.02 4-GXblood test Sensitivity (%) 92.52 85.80 to 96.72 Specificity (%) 92.7185.55 to 97.02 Positive predictive value (%) 93.40 86.87 to 97.30Negative predictive value (%) 91.75 84.39 to 96.37 Hydrogen breath testSensitivity (%) 73.15 63.76 to 81.22 Specificity (%) 85.57 76.97 to91.88 Positive predictive value (%) 84.95 76.03 to 91.52 Negativepredictive value (%) 74.11 64.97 to 81.92 Capillary-blood glucose testSensitivity (%) 69.44 59.84 to 77.95 Specificity (%) 78.35 68.83 to86.07 Positive predictive value (%) 78.13 68.53 to 85.92 Negativepredictive value (%) 69.72 60.19 to 78.16 CI: confidence interval.

The values and percentages of sensitivity (A), specificity (B), positivepredictive value (C) and negative predictive value (D) of the tests(intestinal lactase deficiency diagnostic test described in the presentinvention, hydrogen breath test and capillary-blood glucose test)performed in patients with clinical history suggestive of lactoseintolerance following the administration of the different doses of 4-GXcompared to the determination of intestinal lactase activity in biopsytaken as the gold standard test are shown in Table 7 and in FIG. 1. Allthe values of sensitivity, specificity, positive predictive value andnegative predictive value obtained using the intestinal lactase testwith 4-GX, and both urine and blood tests, are greater than 90%, whichindicates high reliability of any of these three new diagnostic testsusing 4-GX, whether this involves the determination of the concentrationof xylose in plasma or the determination of the amount of xyloseexcreted in urine after two different times.

The best indicator of the high reliability of these diagnostic testsusing the disaccharide 4-GX is to compare the values obtained in thetest of the invention with the values obtained in the hydrogen breathtest and the capillary-blood glucose test, which are the most commonnon-invasive tests currently used in the clinic for the diagnosis ofintestinal lactase deficiency. Table 7 shows that the values forsensitivity, specificity, positive predictive value and negativepredictive value for the hydrogen breath test varied around 73-85% andthe values for the capillary-blood glucose test varied around 69-78%,never reaching the values shown by any of the 4-GX tests, which in allcases were above 90%.

All the statistical parameters of the hydrogen breath test and thecapillary-blood glucose test presented values and ranges (95% confidenceintervals) that were considerably lower than that of any of the 4-GXtests (Table 7). The sensitivity of the hydrogen breath test was 73.2%(percentage of true positives), which is equivalent to saying that thistest presented 26.8% false negatives (percentage of patients that beinghypolactasic according to the measurement of lactase in biopsy were notdetected as such in this test), whereas the sensitivity of the 4-GXtests was 90.7% (4-hour urine), 93.5% (5-hour urine) and 92.5% (blood),that is 9.3%, 6.5% and 7.5% false negatives respectively (FIG. 1A).

The specificity of the hydrogen breath test was 85.6% (percentage oftrue negatives), which is equivalent to saying that this test presented14.4% false positives (percentage of patients testing positive in thistest that are not hypolactasic according to the measurement of lactasein biopsy), whereas the sensitivity of the 4-GX tests was 92.8% (4-hoururine), 91.8% (5-hour urine) and 92.7% (blood), that is 7.2%, 8.2% and7.3% false positives respectively (FIG. 1B).

Equally, the positive predictive value (percentage of true positivescompared to total positives) and the negative predictive value(percentage of true negatives compared to total negatives) were clearlyless with the hydrogen breath test, 85% and 74.1% respectively, comparedto 93.3% (4-hour urine), 92.7%, (5-hour urine) and 93.4% (blood) of thepositive predictive values for the 4-GX tests (FIG. 1C) and 90% (4-hoururine), 92.7% (5-hour urine) and 91.8% (blood) of the negativepredictive values for the 4-GX tests (FIG. 1D).

Also, the positive likelihood ratio (FIG. 1E) obtained for the hydrogenbreath test was 5.068, whereas with the 4-GX test it varied between11.339 and 12.574, which indicates that the probability that a positiveresult obtained by the determination of lactase by biopsy is alsopositive with any of the 4-GX tests is even twice as high as with thehydrogen breath test.

The likelihood ratio of a negative result (FIG. 1F) was 0.314 with thehydrogen breath test, whereas with the 4-GX tests it varied between0.071 and 0.100, which indicates the probability that a positive resultobtained by determination of lactase by biopsy is negative with any ofthe 4-GX tests is even three times less than with the hydrogen breathtest.

The comparison of the data obtained by the various 4-GX tests of theinvention compared to the hydrogen breath test was also made by lookingat the area under the corresponding ROC (Receiver-OperatingCharacteristic) curves, which are curves indicating the accuracy of adiagnostic test. In these curves, a test is considered as optimum whenthe area under the curve approaches the value of 1, at which value thesensitivity and the specificity of the candidate test are both 100%.

In the case of the hydrogen breath test, this area was 0.8501 (FIG. 2),whereas the area obtained with the 4-GX tests was 0.9282 for the case ofthe concentration of xylose excreted in 4-hour urine, 0.9315 for thecase of the concentration of xylose excreted in 5-hour urine and 0.9268for the xylose concentration in blood (FIG. 2). This difference in thearea under the ROC curves is displayed by the marked displacementtowards the right of the ROC curve of the hydrogen breath test comparedto the other curves corresponding to the 4-GX tests of the presentinvention, also showing a statistical significance level of p<0.05compared to the hydrogen breath test.

The sensitivity and specificity of a diagnostic test can also becalculated from ROC curves. The values for sensitivity and specificityof the diagnostic tests of intestinal lactase deficiency by the use of4-GX using the ROC curves were 90.7% and 93.8% respectively for the 4-GXtest and 4-hour urine samples; 93.5% and 91.8% respectively for the 4-GXand 5-hour urine test samples; and 92.5% and 92.7% respectively for the4-GX and blood test samples. These values are almost identical to thosepreviously described for the same parameters [sensitivity of 90.7% and92.8% respectively (4-hour urine), 93.5% and 91.8% respectively (5-hoururine), and 92.5% and 92.7% respectively (blood)] calculated using thecut-off points previously described, which confirms the validity ofthese cut-off points for the diagnosis of patients presenting lactoseintolerance.

As regards the determination of capillary-blood glucose, as shown inFIG. 1, the statistical parameters were of the same order as thoseobtained with the hydrogen breath test and less than those obtained withthe 4-GX tests. The sensitivity of the capillary-blood glucose test was69.4%, i.e. 30.6% were false negatives, whereas the sensitivity of the4-GX tests varied between 90.7% and 93.5%. The specificity of thecapillary-blood glucose test was 78.4%, which indicates that there were21.6% of false positive results, whereas the specificity of the 4-GXtests of the invention varied from 91.8% to 92.8%. The positive andnegative predictive values for the capillary-blood glucose test were78.13% and 69.72% respectively, whereas the 4-GX tests these valuesvaried between 92.66% and 93.40% and from 90.00% to 92.71% respectively.

Lastly, the positive and negative likelihood ratios for thecapillary-blood glucose test were 3.208 and 0.390 respectively, whereasfor the 4-GX tests these parameters varied from 11.339 to 12.689 and0.071 to 0.100 respectively. Therefore, as in the case of the hydrogenbreath test, the values for sensitivity, specificity, positivepredictive value and negative predictive value for the capillary-bloodglucose test and for the hydrogen breath test did not reach the valuesshown with any of the different 4-GX tests, which in all cases werehigher than 90%.

EXAMPLE 8 Analysis of the Discordant Results Obtained Between theIntestinal Biopsy Test and 4-GX Intestinal Lactase Test in Patients witha Clinical History Suggestive of Lactose Intolerance

Once the results from patients presenting clinical history suggestive oflactose intolerance were obtained by the intestinal biopsy technique andthe 4-GX tests, it was observed that there were subjects for whichdiscrepancies in diagnosis were found using the normal limits of eachthe techniques used. Table 8 shows the subjects in which discrepanciesin diagnosis were observed, taking into account the normal limit of thebiopsy test and the normal limits of the 4-GX tests described in thepresent invention. Table 8 also shows the results of the other twodiagnostic tests used in this study, the hydrogen breath test and thecapillary-blood glucose test, and the number of discrepant results shownby each subject between the results obtained by the intestinal biopsytest compared to the other diagnostic tests performed.

TABLE 8 Results obtained in patients where there was a discrepancybetween the values of lactase measured in biopsy and those obtained bythe other tests performed. The limits of normal of each test areincluded, indicating normal as being greater than or equal (≧) or lessthan or equal (≦). The data of each test that was positive arerepresented as dark grey shaded and those that were negative arerepresented as light grey shaded.

As shown in Table 8, discordant results were observed in 19 subjects, 13(68.4%) of which showed discrepancies between the results obtained withthe biopsy test compared to the three 4-GX tests. This discrepancy wasnot unexpected, although it was limited to 6.3% of the total of 205subjects analysed in this study. Although the direct evaluation oflactase activity in a biopsy of the small intestine is generallyconsidered to be the most accurate of the tests available for thediagnosis of hypolactasia, it has many drawbacks as previouslymentioned; apart from being an invasive test, it only indicates theenzyme activity in a specific part or area of the intestine. This partdoes not generally coincide with that of the highest occurrence of thisenzyme (middle jejunum) as it is very difficult to take samples in thisarea. The results obtained by this test are also very variable betweensamplings. All of these factors lead to questioning the measurement oflactase activity in duodenal biopsies as the “gold standard” for thediagnosis of hypolactasia. In this sense, the determination of thelactase activity by measurement of the concentration of xylose excretedin the urine or present in the blood following ingestion of 4-GX wasdemonstrated to reliably reflect the total activity of this enzyme inthe small intestine.

The systematic comparison in all patients of this study of the data forlactase in biopsy with the three 4-GX tests, together with the hydrogenbreath test and the capillary-blood glucose test enabled us to identifybiopsy lactase values in some patients that were clearly discordant withat least 4 of the 5 other tests, the indicate that these lactasemeasurements in biopsy may reasonably be considered to be falsepositives or false negatives, again casting doubt on the use of thistest for the diagnosis of hypolactasia. As shown in Table 8, thediscordance of the biopsy result with at least 4 of the other tests onlyoccurred in one patient (A01), whereas the other results were discordantwith the other 5 tests.

After assigning the above value of false positives considered for eachof the patients included in Table 9 to the data obtained by the biopsymethod, the statistical parameters were recalculated for all of thetests, this time including the data obtained by biopsy, and new ROCcurves were obtained making the comparison of the 4-GX tests with thehydrogen breath test and also with the capillary glucose test.

TABLE 9 Results obtained in patients that showed a discrepancy betweenthe values of lactase measured in biopsy and those obtained by the othertests performed.

F−: False negative F+: False positive

Thus, as shown in Table 9, 11 patients were identified who showeddiscrepancies in the five diagnostic tests (4-GX and 4-hour and 5-hoururine, 4-GX and blood tests, hydrogen breath test and capillary-bloodglucose test) compared to the biopsy test (5.4% of the total of 205subjects analysed in this study). Consequently, the results for subjectsA03, A06, C12, 155 and 158 were considered as false negatives for thebiopsy test and true positives for the other tests, while subjects A27,H02, H12, 106, 107, and 108 were considered as false positives for thebiopsy test and true negatives for the other tests. After correcting thediagnosis obtained from the biopsy test as indicated in Table 9, thereliability parameters of the six tests used in this study for theevaluation of hypolactasia were recalculated in accordance with thefinal criteria established in Table 9, applying the final criterion offalse negative (F-) or false positive (F+) in the biopsy as establishedby the concordance with the 5 other tests. Consequently, the finalnumber of concordant results in all six diagnostic tests and thecriteria for this diagnostic were 102 (95.3%), 103 (96.3%), 106 (99.1%),104 (97.2%), 84 (78.5%) and 80 (74.8%) for the positive results oflactase activity in biopsy, 4-GX and 4-hour urine test, 4-GX and 5-hoururine test, 4-GX and blood test, hydrogen breath test andcapillary-blood glucose test respectively.

The number of negative results in all the tests were 92 (93.9%), 96(98.0%), 95 (96.9%), 95 (96.9%), 89 (90.8%), and 82 (83.7%),respectively. Similarly, new ROC curves were obtained making thecomparison of the 4-GX tests with the hydrogen breath test and thecapillary-blood glucose test.

As shown in Table 10 and in FIG. 3, after correcting the results ofdiagnosis by biopsy, the recalculated values for sensitivity,specificity, positive and negative predictive values for all of thediagnostic tests of intestinal lactase deficiency by the administrationof 4-GX were above 95%, varying between the ranges of 96.00-98.10 forthe 4-hour urine test, 96.94-99.07 for the 5-hour urine test and97.94-98.11 for the blood test. These values were far higher than forany of the other diagnostic tests used in this study, including thevalues obtained by the biopsy test. The values obtained for thestatistical parameters of sensitivity, specificity, positive predictivevalues and negative predictive values were much better than thoseobtained with the hydrogen breath test and those obtained with thecapillary-blood glucose test (see Table 10 and FIG. 3). In this sense,the percentages of false negatives for the 4-GX and 4-hour urine test,5-hour urine test and blood test were 3.74%, 0.93% and 1.89%,respectively (with a sensitivity of 96.26%, 99.07% and 98.11%,respectively), compared to the percentages of false negatives of 4.67%(sensitivity of 95.33%), 21.50% (sensitivity of 78.50%), and 25.23%(sensitivity of 74.77%) obtained for the biopsy test, hydrogen breathtest and capillary-blood glucose test respectively. The positivelikelihood ratios for the 4-GX tests (FIG. 3E) were 2 to 3 times higherthan for the biopsy test (47.168, 32.361, and 47.585 for the 4-GX and4-hour and 5-hour urine and blood tests respectively compared to 15.570for the biopsy test), and 4 to 5 times higher than the hydrogen breathtest (8.548), and 7 to 10 times higher than the capillary-blood glucosetest (4.579). Lastly, the negative likelihood ratios (FIG. 3F) for the4-GX tests were 1.3 to 5 times less than those obtained for the biopsytest (0.038, 0.010, and 0.019 for the 4-GX and 4-hour and 5-hour urineand blood tests respectively compared to 0.050 for the biopsy test), 6to 20 times less than those obtained for the hydrogen breath test(0.237), and 8 to 30 times less than those in the capillary-bloodglucose test (0.302).

TABLE 10 Statistical parameters obtained after correction of the biopsydiagnostic result (five discrepancies in the tests described in theinvention compared to the biopsy test). CI 95% 4-GX test in 4-hour urineSensitivity (%) 96.26 90.70 to 98.97 Specificity (%) 97.96 92.82 to99.75 Positive predictive value (%) 98.10 93.29 to 99.77 Negativepredictive value (%) 96.00 90.07 to 98.90 4-GX test in 5-hour urineSensitivity (%) 99.07 94.90 to 99.98 Specificity (%) 96.94 91.31 to99.36 Positive predictive value (%) 97.25 92.17 to 99.43 Negativepredictive value (%) 98.96 94.33 to 99.97 4-GX blood test Sensitivity(%) 98.11 93.35 to 99.77 Specificity (%) 97.94 92.75 to 99.75 Positivepredictive value (%) 98.11 93.35 to 99.77 Negative predictive value (%)97.94 92.75 to 99.75 Hydrogen breath test Sensitivity (%) 78.50 69.51 to85.86 Specificity (%) 90.82 83.28 to 95.71 Positive predictive value (%)90.32 82.42 to 95.48 Negative predictive value (%) 79.46 70.80 to 86.51Capillary-blood glucose test Sensitivity (%) 74.77 65.45 to 82.67Specificity (%) 83.67 74.84 to 90.37 Positive predictive value (%) 83.3374.35 to 90.16 Negative predictive value (%) 75.23 66.04 to 83.00 Biopsytest Sensitivity (%) 95.33 89.43 to 98.47 Specificity (%) 93.88 87.15 to97.72 Positive predictive value (%) 94.44 88.30 to 97.93 Negativepredictive value (%) 94.85 88.38 to 98.31 CI: confidence interval.

FIG. 4 shows the new ROC curves of the 4-GX and 4-hour urine, 5-hoururine and blood tests respectively compared to the other tests used inthe study, the hydrogen breath test, capillary-blood glucose test andbiopsy test after correcting the diagnosis of the results obtained fromthe biopsy test. The three curves corresponding to the results obtainedwith the three 4-GX tests are displaced far to the upper left corner,compared to the other ROC curves for the other diagnostic testsincluding biopsy. FIG. 4 shows that the areas under the ROC curves forthe 4-GX tests are now close to the ideal value of 1.0 (varying from0.9758 to 0.9894), these values being higher than those obtained for thehydrogen breath test (0.8852, p<0.0001 to 0.0008), capillary-bloodglucose test (0.8512, p<0.0001) and biopsy test (0.9613 but in this casethe p value was not significant with p varying from 0.08 to 0.5110).

As previously mentioned, one of the patients enrolled in the presenttrial (A01) presented discordance between the values obtained by thebiopsy test compared to at least four of the tests described in thepresent invention. This patient was a false negative. Taking this datapoint into account, the statistical parameters of all the tests wererecalculated, corrected by the biopsy diagnostic result and showingdiscrepancies in at least four of the tests performed compared to thebiopsy test (Table 11). The statistical parameters obtained are verysimilar to those shown in Table 10, where the statistical parameters ofall the tests, corrected by the biopsy diagnostic result and showingdiscrepancies in five of the tests performed compared to the biopsytest. Nevertheless, the global conclusions on the reliability parametersof the tests for intestinal lactase activity, either the urine and/orblood tests, by the administration of 4-GX described in the inventionwere unchanged, after taking into account the discrepancies of theresults obtained from these tests compared to the discrepancies of theresults obtained with at least four or five of the other tests used.

TABLE 11 Statistical parameters obtained after correction of the biopsydiagnostic result (four discrepancies in the tests described in theinvention compared to the biopsy test). CI 95% 4-GX test in 4-hour urineSensitivity (%) 97.20 92.02 to 99.42 Specificity (%) 98.98 94.45 to99.97 Positive predictive value (%) 99.05 94.81 to 99.98 Negativepredictive value (%) 97.00 91.48 to 99.38 4-GX test in 5-hour urineSensitivity (%) 100.00 96.61 to 100.00 Specificity (%) 97.96 92.82 to99.75 Positive predictive value (%) 98.17 93.53 to 99.78 Negativepredictive value (%) 100.00 96.23 to 100.00 4-GX blood test Sensitivity(%) 99.06 94.86 to 99.98 Specificity (%) 98.97 94.39 to 99.97 Positivepredictive value (%) 99.06 94.86 to 99.98 Negative predictive value (%)98.97 94.39 to 99.97 Hydrogen breath test Sensitivity (%) 78.50 69.51 to85.86 Specificity (%) 90.82 83.28 to 95.71 Positive predictive value (%)90.32 82.42 to 95.48 Negative predictive value (%) 79.46 70.80 to 86.51Capillary-blood glucose test Sensitivity (%) 74.77 65.45 to 82.67Specificity (%) 83.67 74.84 to 90.37 Positive predictive value (%) 83.3374.35 to 90.16 Negative predictive value (%) 75.23 66.04 to 83.00 Biopsytest Sensitivity (%) 94.39 88.19 to 97.91 Specificity (%) 92.86 85.84 to97.08 Positive predictive value (%) 93.52 87.10 to 97.35 Negativepredictive value (%) 93.81 87.02 to 97.70 CI: confidence interval.

As demonstrated by the data shown in the present invention, the accuracyof the 4-GX and 4-hour and 5-hour urine, and blood tests at doses of 0.5g and 3 g (or 0.45 g and 2.7 g, respectively, in a 4-GX sample including10 wt % water) respectively for the diagnosis of hypolactasia, takinginto account the normal limits described in the present invention andquantified by the sensitivity, specificity, positive predictor value andnegative predictor value parameters as well as the area under the ROCcurves, was significantly better than that shown by the hydrogen breathtest or the capillary-blood glucose test following an overload oflactose.

The reliability of the 4-GX tests was also shown to be better than themeasurement of lactase activity in a sample of intestinal mucosaextracted by biopsy, there not being any other functional test for thediagnosis of hypolactasia with similar reliability, so these new testshave the potential of becoming the gold standard tests for the diagnosisof this disorder. This characteristic together with their beingnon-invasive tests, the absence of reactions in intolerant patients andthe simplicity of conducting the tests makes these new tests into theoptimum functional diagnostic tool for the diagnosis of lactoseintolerance.

The three tests with 4-GX and 4-hour urine, 5-hour urine and blood havevery similar reliability characteristics, so clinical practice canselect any of them depending on the clinical features of the patient. Inthis sense, perhaps the 4-GX test and 5-hour urine shows the highestsensitivity, despite having a slightly lower specificity, so this mightbe the advantage of this test because the risk of a false negativediagnosis may be higher than the risk of a false positive diagnosis.Also, the 4-GX and 5-hour urine test offers better measurement of thetotal amount of excreted xylose in the case, for example, of patientswith a delay in gastric emptying. On the other hand, the 4-GX and bloodtest provides an alternative to the urine test, especially in smallchildren and infants where urine collection can be problematic, and italso requires a shorter fasting time. A combination of both tests, 4-GXand 5-hour urine and 90-minute blood tests would improve the accuracy ofxylose determination in some patients, for example patients with renaldysfunction, that can give incorrectly low values for urinary xylose.

In the prior art, none of the parameters of the amount of 4-GX to beadministered in the test, the time to wait before evaluating intestinallactase activity applied specifically to each test biological fluid:urine or blood (plasma), had been determined in such a way as to enabledistinguishing false positives in such a statistically effective way,that would make the test into a gold standard or reference test for thediagnosis of this disease, better than the other invasive ornon-invasive tests described in the state of the art. The 4-GX and urineor blood test is a safe and innocuous method for the non-invasivediagnosis of hypolactasia with a virtual absence of the most commoninherent abdominal discomforts associated with the use of the hydrogenbreath test and the capillary-blood glucose test when performed inlactose-intolerant subjects. The 4-GX test is simple and only requiresthe determination of xylose excreted in urine or present in blood, whichare routine determinations in clinical laboratories. The excellentresults shown in the present invention and obtained by the use of the4-GX and urine and/or blood tests substantially improve the diagnosis ofhypolactasia compared to the methods currently used, including themeasurement of lactase activity in small intestinal biopsy samples, sothat the test of the invention could be proposed as a new gold standardtest in the diagnostic study of lactose intolerance. Furthermore, usingthe test described in the present invention, it was also possible tocorrectly diagnose and differentiate between individuals with lactoseintolerance due to allergies and those with enzyme deficiencies ofintestinal lactase.

REFERENCES

-   -   Arola, H. (1994) Diagnosis of hypolactasia and lactose        malabsorption. Scand. J. Gastroenterol. 202 (29 Suppl), 22-35.    -   Davidson, G. P., & Robb, T. A. (1985) Value of breath hydrogen        analysis in management of diarrheal illness in childhood:        Comparison with duodenal biopsy. J. Pediatr. Gastroenterol.        Nutr. 4, 381-387.    -   Dawson, D. J., Lobley, R. W., Burrows, P. C., Miller, V. &        Holmes, R. (1986) Lactose digestion by human jejunal biopsies:        the relationship between hydrolysis and absorption. Gut 27,        521-527.    -   ES478590. Procedimiento de obtención de 3-metil-lactosa        utilizable para la evaluación de la lactasa intestinal.    -   ES482073. Mejoras en la patente principal por procedimiento de        obtención de 3-metil-lactosa utilizable para la evaluación de la        lactosa intestinal.    -   ES2023556. Procedimiento de obtención de        4-O-Beta-D-galactopiranosil-D-xilosa utilizable para la        evaluación diagnóstica de la lactasa intestinal.    -   ES2100131. Procedimiento enzimático de obtención de        β-D-galactopiranosil-D-xilosas utilizables para la evaluación        diagnóstica de la lactasa intestinal.    -   ES2182703. Un procedimiento enzimático para obtener        4-O-β-D-galactopiranosil-D-xilosa,        4-O-β-D-galactopiranosil-D-xilosa obtenida de acuerdo con el        procedimiento, composiciones que la contienen y su use en la        evaluación de la lactasa intestinal.    -   ES2208099. Empleo de 4-galactosil-xilosa en humanos para la        evaluación in vivo de lactasa intestinal como prueba diagnóstica        no invasiva de la deficiencia de este enzima.    -   Koetse, H. A., Stellaard, F., Bijleveld, C. M., Elzinga, H.,        Boverhof, R., Van der Meer, R., Vonk, R. J. &        Sauer, P. J. (1999) Non-invasive detection of low intestinal        lactase activity in children by use of a combined ¹³CO₂/H₂        breath Test. Scand. J. Gastroenterol. 34, 35-40.    -   Levitt, M. D. (1969) Production and excretion of hydrogen gas in        man. N. Engl. J. Med. 281, 122-127.    -   Lifshitz, C. H. Bautista, A., Gapalachrishna, G. S., Stuff, J. &        Garza, C. (1985) Absorption and tolerance of lactose in infants        recovering from severe diarrhea. J. Pediatr. Gastroenterol.        Nutr. 4, 942-94845,46.    -   McGill, D. B. & Newcomer, A. D. (1967) Comparison of venous and        capillary blood samples in lactose tolerance testing.        Gastroenterology 53, 371-374.    -   Metz, G., Jenkins, D. J., Peters, T. J., Newman, A. &        Blendis, L. M. (1975) Breath hydrogen as a diagnostic method for        hypolactasia. Lancet 1, 1155-1157.    -   Newcomer, A. D. & McGill, D. B. (1966) Distribution of        disaccharidase activity in the small bowel of normal and        lactase-deficient subjects. Gastroenterology 51, 481-488.    -   Newcomer, A. D., McGill, D. B., Thomas, P. J. &        Hofmann, A. F. (1975) Prospective comparison of indirect methods        for detecting lactase deficiency. N. Engl. J. Med. 293,        1232-1236.    -   Newcomer, A. D., McGill, D. B., Thomas, P. J. &        Hofmann, A. F. (1975) Prospective comparison of indirect methods        for detecting lactase deficiency. N. Engl. J. Med. 293,        1232-1236.    -   Scriber, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs,        B., Kinzler, K. W. and Vogelstein, B. Eds Vol I, pp 1623-1650.        McGraw-Hill, New York.    -   Scriber, C. R., Beaudet, A. L., Sly, W. S., Sasaki, Y., Lio, M.,        Kameda, H,. Ueda, H. & Aoyagi, T. (1970) Measurement of        ¹⁴C-lactose absorption in the diagnosis of lactase        deficiency. J. Lab. Clin. Med. 76, 824-835.    -   Semenza, G. et al. 2001. The Metabolic and Molecular Bases of        Inherited Disease. McGraw-Hill. Vol I, pp 1623-1650.    -   Triadou, N., Bataille, J. & Schmitz, J. (1983) Longitudinal        study of the human intestinal brush border membrane proteins        distribution of the main disaccharidases and peptidases.        Gastroenterology 85, 1326-1332.    -   Valle, D., Childs, B., Kinzler, K. W. and Vogelstein, B. Eds)        Vol I, pp 1623-1650. McGraw-Hill, New York.

1-11. (canceled)
 12. Non-invasive diagnostic method for evaluation ofintestinal lactase deficiency that comprises the stages of: a)Extracting a blood sample from the individual who is the object of thetest after fasting for 8 Hours; b) Administering via the oral route tothe individual who is the object of the test a quantity of4-O-β-D-galactopyranosyl-D-xylose (4-GX) of between 0.125 g and 6 g; c)Extracting a blood sample from this individual at 90 minutes followingthe administration of 4-GX; d) Determining in vitro the concentration ofxylose in the blood sample extracted in step a); e) Determining in vitrothe concentration of xylose in the blood sample extracted in step c); f)Subtracting the values of xylose concentrations obtained in vitro instep e) from those obtained in vitro in step (d); g) Comparing the valueobtained in step f) with a threshold reference value obtained in vitrofrom a population of healthy control individuals subjected to the sameprotocol, below which threshold the subject is considered to besuffering from intestinal lactase deficiency (hypolactasia).
 13. Themethod according to claim 12, wherein the determination of the bloodxylose concentration is performed using plasma.
 14. The method accordingto claim 12, wherein the doses of 4-GX administered are selected from:0.5 g, 1 g, 3 g and 6 g.
 15. The method according to claim 14, whereinthe dose of 4-GX to be administered is 3 g.
 16. The method according toclaim 12, wherein the reference threshold value of the blood xyloseconcentration following administration to the patient of the dose of 0.5g of 4-GX is 0.41 mg/dL.
 17. The method according to claim 12, whereinthe reference threshold value of the blood xylose concentrationfollowing administration to the patient of the dose of 1 g of 4-GX is0.53 mg/dL.
 18. The method according to claim 12, wherein the referencethreshold value of the blood xylose concentration followingadministration to the patient of the dose of 3 g of 4-GX is 0.97 mg/dL.19. The method according to claim 12, wherein the reference thresholdvalue of the blood xylose concentration following administration to thepatient of the dose of 6 g of 4-GX is 1.44 mg/dL.
 20. The methodaccording to claim 12, wherein the comparison that takes place in stepg) determines the degree of intestinal lactase deficiency (hypolactasia)of the individual subjected to the test.